Screening of promoters from metagenomic DNA and their use for the construction of expression vectors.
This study was focused on the screening of valuable genetic resources, such as promoters from metagenome, and describes a promoter trapping system with a bidirectional probe concept, which can select promoters or operons from various biological resources including metagenomic DNA. A pair of reporters, GFP and DsRed, facing the opposite direction without promoters, is an effective system that can function regardless of the direction of inserted promoters. The feasibility of this system was tested for the isolation of constitutively expressed promoters in E. coli from a soil metagenome, resulting in a potential pool of various promoters for practical application. The analyses of structural organization of the trapped genes demonstrated that constitutively expressible promoters in E. coli were broadly distributed within the metagenome, and suggested that some promoters were useful for the construction of expression vectors. Based on these observations, three constitutive promoters were employed in the expression vector system and their potentials for practical application were evaluated in terms of expression level, protein solubility, and effects on host growth.
Han
Sang-Soo
SS
Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwangju 500-757, Korea.
Lee
Jin-Young
JY
Kim
Won-Ho
WH
Shin
Hyun-Jae
HJ
Kim
Geun-Joong
GJ
D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics*; D023281:Genomics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic